Anais da Academia Brasileira de Ciências (2005) 77(3): 549-579 (Annals of the Brazilian Academy of Sciences) ISSN 0001-3765 www.scielo.br/aabc History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience JOSÉ I. BALDANI and VERA L.D. BALDANI Embrapa Agrobiologia, BR465, Km 07, 23851-970 Seropédica, Rio de Janeiro, Brasil Manuscript received on December 27, 2004; accepted for publication on April 6, 2005; presented by Eurípedes Malavolta ABSTRACT This review covers the history on Biological Nitrogen Fixation (BNF) in Graminaceous plants grown in Brazil, and describes research progress made over the last 40 years, most of which was coordinated by Johanna Döbereiner. One notable accomplishment during this period was the discovery of several nitrogen-fixing bac- teria such as the rhizospheric (Beijerinckia fluminensis and Azotobacter paspali), associative (Azospirillum lipoferum, A. brasilense, A. amazonense) and the endophytic (Herbaspirillum seropedicae, H. rubrisub- albicans, Gluconacetobacter diazotrophicus, Burkholderia brasilensis and B. tropica). The role of these diazotrophs in association with grasses, mainly with cereal plants, has been studied and a lot of progress has been achieved in the ecological, physiological, biochemical, and genetic aspects. The mechanisms of colonization and infection of the plant tissues are better understood, and the BNF contribution to the soil/plant system has been determined. Inoculation studies with diazotrophs showed that endophytic bacteria have a much higher BNF contribution potential than associative diazotrophs. In addition, it was found that the plant genotype influences the plant/bacteria association. Recent data suggest that more studies should be conducted on the endophytic association to strengthen the BNF potential. The ongoing genome sequencing programs: RIOGENE (Gluconacetobacter diazotrophicus) and GENOPAR (Herbaspirillum seropedicae) reflect the commitment to the BNF study in Brazil and should allow the country to continue in the forefront of research related to the BNF process in Graminaceous plants. Key words: bacterial endophytes, diazotrophs, semi-solid N-free medium, inoculation, cereals, grass plants. INTRODUCTION on the occurrence of Azotobacter in acid soils of the “Baixada fluminense” (Döbereiner 1953). These Research on BNF with grasses in Brazil was initiated studies gained visibility with the discovery of two by Johanna Döbereiner when she joined the research new nitrogen-fixing bacteria associated with the rhi- team at the National Center of Education and Agri- zosphere of some gramineous plants: Beijerinckia cultural Research of the Ministry of Agriculture, lo- fluminensis with sugarcane (Döbereiner and Ruschel cated at Km 47 in the fifties. The first studies were 1958) and Azotobacter paspali with Paspalum no- Dedicated to the memory of Dr. Johanna Döbereiner by two of tatum cv. batatais (Döbereiner 1966). In the sev- her disciples who learned through working with her that research enties, a significant advance in the area of BNF in could be done with simplicity, perseverance, honesty, ethics and grasses was the introduction of the acetylene reduc- sagacity. tion method. Almost at the same time, the semi- Correspondence to: José Ivo Baldani E-mail: [email protected] solid NFb medium that replicated the oxygen level An Acad Bras Cienc (2005) 77 (3) 550 JOSÉ I. BALDANI and VERA L.D. BALDANI found in soil niches was developed to isolate mi- et al. 1997a). New nitrogen-fixing bacteria were croaerophilic nitrogen-fixing bacteria associated identified: Herbaspirillum rubrisubalbicans (Bal- with plant roots. This medium allowed the isolation dani et al. 1996), Herbaspirillum frisingense (Kirch- of two new species of Azospirillum: A. lipoferum hof et al. 2001), Azospirillum doebereinerae (Eck- and A. brasilense. This marked the beginning of ert et al. 2001), Gluconacetobacter johannae and BNF research in grasses in Brazil as well in other Gluconacetobacter azotocaptans (Fuentes-Ramírez countries. The research concentrated on several ar- et al. 2001) and Burkholderia tropica (Reis et al. eas of the interaction between plants and bacteria 2004). including the microorganisms themselves. A new This review aims to rescue the history of BNF species of Azospirillum named A. amazonense studies with grasses in Brazil. These studies were (Magalhães et al. 1983) was isolated and identified mainly coordinated by Johanna Döbereiner and using semi-solid LGI medium, derived from modifi- through her efforts this line of investigation was es- cation of the pH and carbon source of NFb medium tablished in several parts of the World. In addition, (Baldani et al. 1984). it presents recent advances in the area. Several lines of research focusing on agricul- tural applications were developed. Hormonal ef- Free-living and Rhizospheric fects, nitrogen assimilation, biological nitrogen fix- Nitrogen-fixing Bacteria ation and even negative responses were frequently Azotobacter chroococcum observed (Boddey and Döbereiner 1982). During These studies were mostly done between 1950 and this period, other groups working on BNF with non- 1970 when very little was known about the occur- leguminous plants were established in Brazil. Cur- rence of these bacteria in tropical soils. In the first rently, besides the Embrapa Agrobiologia (Km 47) paper on the subject, Döbereiner (1953) observed a team there is groups in the States of Paraná, Rio very frequent occurrence of Azotobacter chroococ- Grande do Sul, Rio de Janeiro, Minas Gerais, Goiás, cum in 22 out of 27 acid soil samples collected in Ceará and Distrito Federal. the “Baixada fluminense”. Research on the colonization of plant tissues by diazotrophic bacteria received a lot of attention from 1985 to 1990 and consequently some aspects of Beijerinckia fluminensis the plant-bacteria interaction began to be elucidated. The occurrence of nitrogen-fixing bacteria of the Two new nitrogen-fixing bacteria able to colonize genus Beijerinckia was mentioned for the first time the interior of plant tissues were found: Herbaspiril- in Brazil and it was demonstrated that the size of lum seropedicae, was isolated from plants of maize, the populations was related to vegetation, physical, sorghum and rice (Baldani et al. 1986a) and Glu- and chemical characteristics of the soil (Döbere- conacetobacter diazotrophicus (synon. Acetobacter iner and Castro 1955). Additional studies on the diazotrophicus) was isolated from sugarcane plants occurrence of this genus in soil of several Brazil- (Cavalcante and Döbereiner 1988). ian States (Rio de Janeiro, São Paulo, Pernambuco This intimate interaction between macro and and Paraná) led to the description of a new species micro symbionts modified the concept known as of Beijerinckia named B. fluminensis (Döbereiner associative and Döbereiner (1992a) introduced the and Ruschel 1958). Analysis of 158 samples col- endophyte concept to the field of BNF. From this lected in different regions of Brazil showed that this point on, a new area within BNF was established species occurred predominantly in soils where sug- which led to great advances in the understanding of arcane was cultivated (Döbereiner 1959a) and a di- physiology, ecology and genetics as well as in the rect influence of the plant on the development of the interaction of the bacteria with the plant (Baldani bacteria was suggested (Döbereiner 1959b). Addi- An Acad Bras Cienc (2005) 77 (3) HISTORY ON BNF IN GRAMINACEOUS PLANTS GROWN IN BRAZIL 551 tional studies showed that roots as well as leaves this grass showed that Beijerinckia and Azotobacter and stems had a positive influence on Beijerinckia were the predominant bacteria in the rhizosphere, populations. This was influenced by the exudation even with plants grown in acid soils (Ruschel and of substances into the soil by the roots during rain- Döbereiner 1965). The use of silica gel plates, con- fall (Döbereiner and Alvahydo 1959). Other stud- taining Winogradsky salts and calcium citrate as a ies involving the roots showed that the population carbon source, inoculated with rhizoplane or rhizo- of Beijerinckia was much more pronounced in the sphere soil of Paspalum notatum, led to the isolation rhizoplane region (refers to the soil adherent to the of a new species of Azotobacter, named Azotobac- root surface) than in the rhizosphere. In addition, ter paspali (Döbereiner 1966). This name was later it was shown that removal of the aerial part of the changed to Azorhizophilus paspali (Thompson and plant significantly reduced the population of bacte- Skerman 1981). The high frequency of this bac- ria in both the rhizoplane and rhizosphere regions terium in the rhizoplane of P. notatum (75 out of (Döbereiner 1961). Rice plants grown in green- 76 samples) and in two of three samples of P. plica- house and inoculated with Beijerinckia, showed the tum, as well as its complete absence in 81 rhizoplane establishment of the bacteria as well as an increase samples of other plants including 5 species of Pas- in the yield (Döbereiner and Ruschel 1961). How- palum, led to the suggestion that the observed speci- ever, these types of experiments were not continued. ficity could represent an intermediate stage between Studies on the occurrence of Beijerinckia and Azoto- the legume symbiosis and the non-symbiotic nitro- bacter were extended to other forage grasses and the gen fixation (Döbereiner 1970). Additional stud- results showed that Beijerinckia appears mainly in ies concerning the dependency